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This study was to develop the test kit for detecting antibiotic residuals in raw milk, using the principle of yogurt producing. Water samples were collected from three hot springs located in Ratchaburi and Kanchanaburi province. After purification, species of microbes were identified by Polymerase Chain Reaction (PCR). DNA sequencing presented about 97% similarity of all samples to Geobacillus stearothermophilus. Three isolates were conducted Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) that the data from MIC and MBC used to support the selection for further steps. The conditions of stock preparation were 64°C of incubation for 18-20 hours produced the maximum spore of 107 CFU/mL and the minimum spore of 105 CFU/mL. The test kit consisted of 106 CFU/mL of G. stearothermophilus spore adjusted pH 9.5 and used bromocresol purple as a pH indicator could detect antibiotic residual at 5 hours of 64 ͦ C of incubation. Detection limits of Amoxicillin, Penicillin, Ampicillin, Ceftiofur, and Oxytetracycline were 4, 8, 8, 100, and 200 µg/l, respectively. The detection limits from the developed test were close to the standard of Codex Alimentarius International Food Standards and Australian Pesticides and Veterinary Medicines Authority.
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